Thrombolytic composition

ABSTRACT

The invention disclosed relates to thrombolytic combination therapy by the administration of an agent capable of activating native plasminogen, and a non-native plasminogen analogue which is cleavable by native thrombin to generate plasmin activity.

This invention relates to products and methods for improved thrombolytictreatment of patients having, or at risk of developing, a thrombus orthrombi. In particular, the invention relates to thrombolyticcombination therapy by the administration of an agent capable ofactivating native plasminogen, and a non-native plasminogen analoguewhich is cleavable by native thrombin to generate plasmin activity.

BACKGROUND OF THE INVENTION

Plasminogen Activators

Agents capable of activating native plasminogen are known and are in usein thrombolytic therapy, are in clinical trial or have been proposed forthat purpose. Examples of such compounds include tissue-type plasminogenactivator (tPA), urokinase, streptokinase, and desmodus salivaryplasminogen activator (DSPA). Intravenous treatment with tPA andstreptokinase has been successful in reducing mortality from acutemyocardial infarction.

Despite this success, it is widely recognised that thrombolysis hasmajor limitations which are due to the shortcomings of the agentsavailable (reviewed by Marder and Sherry, New England Journal ofMedicine 1989, 318: 1513-1520). For example, for tPA these are its rapidelimination from the body and its relative lack of thrombus specificityat clinically useful doses. The rapid clearance of tPA means that alarge quantity of the protein has to be given by intravenous infusion,delaying the start of therapy until the patient is admitted to hospital.A further problem associated with the rapid clearance of tPA is thatreocclusion of the reperfused blood vessel commonly occurs when tPAadministration is stopped. Finally, the relative lack of thrombusspecificity of therapeutic doses of tPA produces a risk of haemorrhage.Risk of haemorrhage is a problem common to current thrombolytics becausethey are not thrombus specific at clinically useful doses and theyactivate plasminogen to produce plasmin in the general circulation.

Urokinase has a similar rapid plasma clearance and also requiresadministration by continuous infusion.

Many attempts have been made to increase the half-life of tPA toovercome its rapid clearance, but tPA variants in clinical developmentappear to be cleared only 3-5 times more slowly than tPA (Thromb.Haemostas. 66:569-574, 1991; Thromb. Haemostas. 70:307-312, 1993); theplasma half-life of tPA is around five minutes in man (Bounameaux et al.in: "Contemporary issues in Haemostasis and Thrombosis" vol 1 p5-91,1985. Collen et al. eds, Churchill Livingstone). These tPA variants maywell allow bolus administration and/or dose reduction but they areunlikely to remain in circulation long enough to be effective inpreventing reocclusion. They are also unlikely to reduce thehaemorrhagic risk significantly, and it may increase due to persistencein the circulation.

Another approach to improving the clinical efficacy of plasminogenactivators is via adjunctive therapy with anticoagulant and antiplateletagents. There has been limited success in improving the therapeuticefficiency of tPA with the adjunct use of heparin and aspirin. However,such a therapeutic regime still has limitations of a 15-20% rate offailure to recanalize the infarct-related artery and by a reocclusionrate of 5-10%. As a result, the reinfarction rate at 30 days is 4-6%,cardiogenic shock and congestive heart failure occur in 5-7% and 15-17%of patients, respectively, and mortality is in the range of 5-8% (Am. J.Cardiol. 75:7-13 1995). Both the initial failure of thrombolysis andreocclusion despite the use of heparin and aspirin are thought to be theresult of ongoing thrombin activity. Hence, more potent and selectiveagents are under investigation. Hirudin is a potent inhibitor ofthrombin which, in animal models has been shown to be superior toheparin in decreasing platelet deposition and thrombus formation(Circulation 79: 657-665, 1989) and in models of coronary thrombosis hasbeen shown to both speed thrombolysis and decrease reocclusion(Circulation 83:1048-1056 and Circulation Research 70:829-834, 1992).U.S. Pat. No. 4,944,943 discloses the use of thrombolytic agents such astPA and antithrombotic agents, hirudin being the only suggested example,for adjunctive therapy. U.S. Pat. No. 5,126,134 discloses the use ofplasminogen activator such as tPA and hirudin, for adjunctive therapy.However, three clinical trials investigating hirudin as an adjunct totPA were stopped in 1994 because of a high incidence of bleeding(Circulation 90:1624-30, 1631-37 and 1638-42, 1994). In addition, thesetrials investigated a higher heparin dose than the previous norm andfound that it too was associated with an increased haemorrhageincidence. Therefore it appears that intensification of adjunctantithrombotic therapy using agents which inhibit thrombin activityresults in an unacceptable increase in bleeding.

Thrombin Activatable Plasminogen Mutants

Patent publications WO 91/09118 and WO 94/10318 (both BritishBio-technology Ltd) describe a new approach to the treatment ofthrombotic conditions, based on the use of mutants of plasminogen whichare activated to generate plasmin or plasmin-like activity by cleavageby an enzyme which is itself involved in the natural clotting cascade,particularly thrombin. One advantage of such agents lies in theirthrombus specificity. Since activation occurs on exposure to thrombin,which is particularly concentrated at the site of the thrombus,thrombolytic plasmin activity is generated where it is required, therebyreducing the risk of systemic activation and haemorrhage. It is to beexpected that such compounds will have an antithrombotic effect also,because the tendency to thrombus formation will be counteracted by thethrombolytic plasmin activity generated by thrombin cleavage of theagent at that site. Natural Glu-plasminogen has a plasma half-life of2.2 days (Thromb. Haemostas. 43:77-89 1980), thus prolonged persistencein the circulation confers a further advantage on thrombin-activatableplasminogen which complements the thrombus selectivity of its action.

The potency of the thrombin activatable plasminogen mutants of WO91/09118 and WO 94/10318 may be enhanced by the further mutationdisclosed in WO 94/03614 (British Bio-technology Ltd) which is designedto introduce resistance to antiplasmin, the cognate serine proteaseinhibitor of plasmin.

BRIEF DESCRIPTION OF THE INVENTION

This invention is based on the finding of the present inventors thatthrombin activatable plasminogen analogues of the kind referred to aboveare capable of increasing the thrombolytic activity of plasminogenactivating agents such as tPA without significantly increasing systemicplasmin production and therefore the risk of haemorrhage. This findingmakes available a new more effective thrombolytic therapy by permittingbolus administration of the plasminogen activator and thus the earlierstart of therapy; by reducing the effective dose of the plasminogenactivator required, thereby reducing the haemorrhagic risk; and byreducing the incidence of reocclusion of opened vessels by reformingthrombi.

DETAILED DESCRIPTION OF THE INVENTION

According to the present invention there is provided a productcontaining a plasminogen activator and a thrombin-activatableplasminogen analogue, for simultaneous, separate or sequential use inthrombolytic therapy.

The invention also includes a method of treating a patient in need ofthrombolytic or antithrombotic therapy, which comprises thesimultaneous, separate or sequential intravenous administration ofeffective amounts of a plasminogen activator and a thrombin-activatableplasminogen analogue.

According to another aspect of the invention there is provided for theuse of a thrombin-activatable plasminogen analogue in the preparation ofan agent for:

a) treating a patient undergoing thrombolytic therapy with a plasminogenactivator; and/or

b) reducing the effective dose of a plasminogen activator required for apatient undergoing thrombolytic therapy with said plasminogen activator;and/or

c) reducing the risk of haemorrhage in patients undergoing thrombolytictherapy with a plasminogen activator.

Such adjunct antithrombotic therapy can be used for the treatment ofacute vascular diseases such as: myocardial infarct, stroke, unstableangina, pulmonary embolism, deep vein thrombosis, peripheral arterialocclusion, extracorporeal circulation, arteriovenous shunts and othervenous thromboses to produce accelerated removal of pathologicalthrombus.

By the term "plasminogen activator" is meant a compound which activatesplasminogen to generate plasmin-type thrombolytic activity. Examples ofsuch compounds include those which directly cleave plasminogen togenerate plasmin, such as tPA, DSPA, urokinase, and mutants of theseproteins having sequence changes which do not destroy their ability tocleave plasminogen to release plasmin, as well as compounds whichindirectly activate plasminogen, such as streptokinase, which forms acomplex with plasminogen causing conformational changes which allow thecomplex to cleave plasminogen molecules to release plasmin.

By the term "thrombin-activatable plasminogen analogue" is meant amolecule differing from wild type plasminogen by having sequence changeswhich render the molecule cleavable by thrombin to release plasmin or amolecule substantially homologous to plasmin which retains plasmin-typeactivity. Thrombin-activatable plasminogen analogues are inefficientlyactivated by plasminogen activators when compared to naturalplasminogen. The thrombin-activatable plasminogen analogues may alsohave sequence changes relative to wild type plasminogen which eitherhave no effect on the thrombin-cleavability of the molecules and theplasmin activity of the cleavage product, or confer an additionalbenefit on the molecules while retaining thrombin-cleavability and theplasmin activity of the cleavage product, for example mutations whichconfer antiplasmin resistance as described in WO 94/03614.

Thrombin-activatable plasminogen analogues are disclosed in the BritishBio-technology patent publications referred to above. The mutationswhich render the molecule cleavable by thrombin to generate plasminactivity, are preferably at or near the position in the moleculecorresponding to the natural cleavage site of wild type plasminogen. Inthat connection, plasminogen was numbered as a result of the proteinsequencing studies of Sottrup-Jensen et al. (in: Atlas of ProteinSequence and Structure (Dayhoff, M.O., ed.) 5 suppl. 3, p.95 (1978))which indicated that plasminogen was a 790 amino acid protein and thatthe site of cleavage was the Arg(560)-Val(561) peptide bond. However, asuitable plasminogen cDNA useful as an intermediate in the synthesis ofthrombin-activatable plasminogen analogues is that isolated by Forsgrenet al (FEBS Letters 213:254-260 (1987)), which codes for a 791 residueprotein with an extra lie at position 65. In this specification, thenumbering of the amino acids in plasminogen corresponds to that of theForsgren cDNA.

The presently preferred plasminogen activator for use in the presentinvention is tPA, but "second generation tPAs" such as that known as BM06.022 (Martin. U. et al. Thromb. Haemostas. 66:569-574, 1991) or thatknown as T103N.KHRR296-299AAAA (Paoni N.F. et al. Thromb. Haemostas.70:307-312, 1993) may become preferred as and when approved for humantherapy by the regulatory authorities.

Presently preferred thrombin-activatable plasminogen analogues for usein the present invention .include those disclosed in WO 94/10318, whichhave a cleavage site sequence P4-P3-Pro-Arg-P1'-P2' where P3 is a basicamino acid residue, P4 is a hydrophobic amino acid residue and each ofPI' and P2' is independently a non-acidic amino acid residue, said sitebeing cleavable by thrombin between Arg and P1'. A particularlypreferred thrombin-activatable plasminogen analogue for use in theinvention is BB-10153, as described in example 2 of WO 94/10318, whichis a plasminogen analogue in which, relative to wild type plasminogen,the amino acid residues Pro(559), Gly(560) are replaced byThr-Thr-Lys-Ile-Lys-Pro, and Va1(562) is replaced by lie to produce acleavage Iccp cleavable by thrombin; and two additional amino-acidsubstitutions Glu606 to Lys and Glu623 to Lys serve to impair binding ofα2-antiplasmin.

In the products of the invention, the plasminogen activator and thethrombin-activatable plasminogen analogue are, for reasons of regulatoryrequirements and/or product quality control, preferably packagedseparately in unit dose form, particularly for separate intravenousbolus injection. Standard injectable protein formulation technologyenables the preparation of suitable unit dose formulations. The unitdose concentration of tPA will generally be in the range of 0.05-1.5mg/kg body weight, preferably 0.1-0.5 mg/kg body weight, and the unitdose concentration of thrombin-activatable plasminogen analogue willgenerally be in the range of 0.1-5 mg/kg body weight, preferably 0.5-3mg/kg body weight, more preferably 2 mg/kg body weight.

The maximum thrombin-activatable plasminogen analogue BB-10153 dose forman will generally be no more than 5 mg/kg.

As an alternative, the products of the invention may contain single unitdoses of a mixture of the plasminogen activator and thethrombin-activatable plasminogen analogue, providing the formulationtechnology employed for its preparation ensures satisfactory stabilityand preservation of potency of the two components.

For the treatment of human patients, as an individual therapeutic, tPAwas originally given as a 100 mg dose by intravenous infusion over 3hours. This has now been superseded by an accelerated regime comprisingan intravenous bolus dose of 15 mg followed by an intravenous infusionof 0.75 mg/kg of body weight over a 30 minute period (maximum 50 mg) andthen an infusion of 0.5 mg/kg over 60 minutes (maximum 35 mg) (J. Am.Coil. Cardiol. 14:1566-1569, 1989 and N. Eng. J. Med. 329:673-682,1993). tPA is not effective as a single i.v. bolus although it hasrecently been reported to be effective as two 50 mg i.v. bolusinjections (J. Am. Coil. Cardiol.23:6-10, 1994). Reteplase (BM 06.022),long-acting tPA variant, has been administered as a double intravenousbolus of 10 million units (MU) followed 30 minutes later by 5 MU (Am. J.Cardiol. 72:518-524, 1993).

Streptokinase is administered as a 1.5 million unit dose by intravenousinfusion over 1 hour.

The effective dosages of the plasminogen activator and thrombinactivatable plasminogen analogues for administration can, based on theknowledge of current dosages and the potential reduction in tPA dosepossible, be determined by the physician.

The inventors have discovered, from animal studies, that a 3-fold ormore reduction in tPA dose is generally possible with the adjunctthrombin-activatable plasminogen analogue BB-10153, both agents given asan i.v. bolus. A similar reduction would be expected using otherplasminogen activators. Thrombin-activatable plasminogen analogueadjunct administration may therefore allow the plasminogen activatoragents to be given as a single bolus.

The maximum tPA dose in any current regime is 100 mg. Byco-administration of a thrombin activatable plasminogen analogue theeffective total tPA dose may be reduced to 80 mg, and preferably 70 mg,60 mg, 50 mg, 40 mg, 35 mg, 30 mg 20 mg or 10 mg.

By co-administration of a thrombin activatable plasminogen analogue theeffective total Reteplase tPA dose may be reduced to 12 MU, andpreferably 10 MU, 8 MU, 7 MU, 6 MU, 5 MU, 4 MU, 3 MU, 2 MU or 1.5 MU. Asimilar total dose reduction may be achieved for the other "secondgeneration tPAs" including that known as T103N .KH RR296-299AAAA.

By co-administration of a thrombin activatable plasminogen analogue theeffective total streptokinase dose may be reduced to 1.2 MU, andpreferably 1 MU, 0.8 MU, 0.7 MU, 0.6 MU, 0.5 MU, 0.4 MU, 0.3 MU, 0.2 MUor 0.15 MU.

Where the plasminogen activator and the thrombin-activatable plasminogenanalogue are administered separately each by intravenous bolusinjection, the order of administration is not critical. It may bepreferable to administer the plasminogen activator first, followed bythe thrombin-activatable plasminogen analogue.

Where the thrombin-activatable plasminogen analogue and tPA agents areadministered separately, it is contemplated that the the first agent maybe administered 1-30 minutes, preferably about 10 minutes, morepreferably about 5 minutes, after administration of the first agent.

Administration of the plasminogen activator and the thrombin-activatableplasminogen analogue may also be by intravenous infusion, either fromseparate sources of the two components or (again providing theformulation technology employed for its preparation ensures satisfactorystability and preservation of potency of the two components) from asingle source of the mixed components.

Simultaneous administration may be affected using a devise capable ofadministering two substances simultaneously. Alternatively, simultaneousadministration is also contemplated to include the administration ofeither the thrombin-activatable plasminogen analogue or tPA first, andthe other component may be administered immediately afterwards."immediately" in this context meaning any period of time up to oneminute after administration of the first component.

Alternatively, administration of either the plasminogen activator or thethrombin-activatable plasminogen analogue may be by intravenous infusionand the other by intravenous bolus injection.

One embodiment of the present invention is illustrated in the followingnon-limiting example.

EXAMPLE

Preparation of a thrombin-activatable plasminogen analogue

The design, construction, expression and purification of thethrombin-activatable plasminogen analogue BB-10153 is described in WO94/10318.

BB10153 is a plasminogen analogue in which the amino acid residuesPro(559), Gly(560) are replaced by Thr-Thr-Lys-Ile-Lys-Pro, and Val(562)is replaced by lie to produce a cleavage loop cleavable by thrombin; twoadditional amino-acid substitutions Glu606 to Lys and Glu623 to Lysserve to impair binding of α2-antiplasmin.

Biological testing of Adjunctive effects of Intravenous tPA and BB-10153

The thrombolytic activities of tPA and thrombin activatable plasminogenBB-10153 were measured in a rabbit arterial thrombosis model in whichthrombus formation in the femoral artery is induced by insertion of acoil of copper wire (copper is a potent stimulus for thrombusformation). Blood flow was monitored just distal to the coil by anultrasonic flow probe and meter.

Male New Zealand White rabbits, 2.5-3.0 kg, were anaesthetised byintravenous injection (ear vein) with a loading dose of 35 mg/kg of a17.5 mg/ml pentobarbitone solution. Once the venous cannula was in placepentobarbitone was infused throughout the experiment at 18 mg/kg/hr(infusion rate of 2 ml/hr). The trachea of the anaesthetised rabbit wascannulated to allow the animal to be artificially respired. The endtidal CO2 was set to 6% and the inspired air was enriched with oxygen.Both jugular veins were cannulated, one for the anaesthetic infusion andthe other for bolus doses of test compounds. The left carotid artery wascannulated to record blood pressure and heart rate.

The femoral artery from both limbs was dissected free from surroundingtissue making sure no side branches were tied. A laparotomy wasperformed and the peritoneum opened above the position where the aortabifurcates into the lilac arteries. A flow probe (2SB Transonic) wasplaced on the left femoral artery distal to the lateral circumflexartery and superficial epigastric artery (approx 2-3 cm distal to theepigastric). Then approx. 5 ml of bicarbonate was given to correct anyacidosis. A blood sample was taken to check the pH (7.35-7.45) and theblood gases pO₂ (100-120 mmHg) and pCO2(35-45 mmHg). A copper coil madefrom 10 turns of 0.5 mm diameter wire wrapped around a 21 gauge needlewas used to induce a thrombus. The coil was positioned in the leftfemoral artery just distal to the lateral circumflex artery and proximalto the flow probe by cannulation of the right femoral artery. Branchesbetween the coil and probe were tied. A cannula was used to advance thecoil up into the right lilac artery and then into the aorta where theblood carries it down to the left lilac artery to the femoral artery.The coil is finally positioned using forceps. Blood flow was monitoredjust distal to the coil in the femoral artery by an ultrasonic flowprobe and meter. Blood flow through the left femoral artery ceasedwithin 5 minutes of placing the coil in the vessel and treatment started30 minutes after cessation of flow. For the combined treatment, BB-10153was given first as an intravenous bolus injection and was followed 5minutes later by tPA, also given as a bolus. 1.0 ml blood samples weretaken from a carotid artery for analysis before and at 0.5, 1, 2, 3 and4 hours after drug administration. The samples were anticoagulated with3.8% w/v trisodium citrate (1 part in 10). The samples were spun at14000 rev/min for 10 minutes and the plasma was then used forhaemostatic protein determinations (see Methods).

Thrombolytic activity of tPA

tPA (Actilyse, purchased from Boehringer Ingelheim) at 3 mg/kg producedprolonged reperfusion in 4/6 animals and short periods of reperfusion inthe remaining 2. tPA at 1 mg/kg induced significant periods ofreperfusion in 2/8 animals but there was no reperfusion in the other 6.tPA at 0.3 mg/kg did not induce any significant reperfusion.

Thrombolytic activity of BB-101.53

BB-10153 did not induce reperfusion of the occluded femoral artery whenadministered at doses up to 4 mg/kg.

Thrombolytic activity of combined treatment with tPA and BB-10153

The thrombolytic potency of tPA was increased significantly byco-administration of BB-1 0153. The combination of 1 mg/kg tPA and 2mg/kg BB-10153 induced prolonged reperfusion in 4/6 animals, asignificant but shorter reperfusion in 1 animal and several shortperiods of flow in 1. This pattern was similar to that produced by 3mg/kg tPA. The combination of 0.3 mg/kg tPA and 2 mg/kg BB10153 inducedsubstantial periods of flow in 3/4 animals,

Effect of tPA and BB-10153 on plasma α2-antiplasmin and fibrinogenlevels

Increasing the thrombolytic potency of tPA by co-administration ofBB-10153 was not accompanied by a significant increase in the systemicgeneration of plasmin. tPA at 3 mg/kg produced marked systemic plasmingeneration as evidenced by reduction of the plasma α2-antipiasmin(α2-PI) level to less than 20% of control and reduction of the plasmafibrinogen level to approximately 25% of control. tPA at 1 mg/kg reducedthe α2-PI and fibrinogen levels to around 60% and 75% of controlrespectively. BB-10153 at 2 mg/kg had no effect on plasma α2-PI andfibrinogen levels. The combination of 1 mg/kg tPA and 2 mg/kg BB-10153produced only a minor increase in systemic plasmin activity compared tothat produced by 1 mg/kg tPA alone.

tPA at 0.3 mg/kg had little effect on the α2-PI and fibrinogen levelsand this was unchanged with the thrombolytic combination of 0.3 mg/kgtPA and 2 mg/kg BB10153.

METHODS

Haemostatic proteins

All determinations were performed on an ACL300R coagulometer fromInstrumentation Laboratories; the coagulometer was supplied with a PCand relevant software (Research software). All reagents and kits for thecoagulation tests were supplied by Instrumentation Laboratories,

α2-antiplasmin the assay was performed using the standard kit suppliedfor the coagulometer and the default settings for the antiplasmin test.

Fibrinogen

A modification of the Clauss method was used (Clauss. A, Acta Haematol.17:237 1957) A calibration curve was created using calibration plasma(the reciprocal of the fibrinogen concentration vs time to clot). Plasmafor the calibration curves was obtained from InstrumentationLaboratories (IL). The concentrations were 2.95, 1.475, 0.98, 0.7375,0.59 and 0.295 g/l (dilutions from stock are stock, 1:2, 1:3, 1:4, 1:5and 1:10). Fibrinogen in the test blood samples was determined from thecalibration curve using the thrombin time assay results (see below).Analysis of the data was carried out on a PC using the research softwaresupplied by IL to go with the ACL 300R coagulometer.

Thrombin time

A standard kit was used with the modification that thrombin was made upwith 100 mmol CaCl₂ to a conc of 3 NIHU/ml. The assay was performedusing research mode (i.e clot cycle 1) settings--volumes of sample andreagent 75 μl, activation time 180 sec, inter ramp interval 1 sec,acquisition time 300 sec, speed 1200 rpm. Analysis using the Pathmatt.def (S/RX100.S4.S4.threshold (3.50)).

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 1                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 814 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       MetGluHisLysGluValValLeuLeuLeuLeuLeuPheLeuLysSer                              19- 15-10-5                                                                   GlyGlnGlyGluProLeuAspAspTyrValAsnThrGlnGlyAlaSer                              1510                                                                          LeuPheSerValThrLysLysGlnLeuGlyAlaGlySerIleGluGlu                              152025                                                                        CysAlaAlaLysCysGluGluAspGluGluPheThrCysArgAlaPhe                              30354045                                                                      GlnTyrHisSerLysGluGlnGlnCysValIleMetAlaGluAsnArg                              505560                                                                        LysSerSerIleIleIleArgMetArgAspValValLeuPheGluLys                              657075                                                                        LysValTyrLeuSerGluCysLysThrGlyAsnGlyLysAsnTyrArg                              808590                                                                        GlyThrMetSerLysThrLysAsnGlyIleThrCysGlnLysTrpSer                              95100105                                                                      SerThrSerProHisArgProArgPheSerProAlaThrHisProSer                              110115120125                                                                  GluGlyLeuGluGluAsnTyrCysArgAsnProAspAsnAspProGln                              130135140                                                                     GlyProTrpCysTyrThrThrAspProGluLysArgTyrAspTyrCys                              145150155                                                                     AspIleLeuGluCysGluGluGluCysMetHisCysSerGlyGluAsn                              160165170                                                                     TyrAspGlyLysIleSerLysThrMetSerGlyLeuGluCysGlnAla                              175180185                                                                     TrpAspSerGlnSerProHisAlaHisGlyTyrIleProSerLysPhe                              190195200205                                                                  ProAsnLysAsnLeuLysLysAsnTyrCysArgAsnProAspArgGlu                              210215220                                                                     LeuArgProTrpCysPheThrThrAspProAsnLysArgTrpGluLeu                              225230235                                                                     CysAspIleProArgCysThrThrProProProSerSerGlyProThr                              240245250                                                                     TyrGlnCysLeuLysGlyThrGlyGluAsnTyrArgGlyAsnValAla                              255260265                                                                     ValThrValSerGlyHisThrCysGlnHisTrpSerAlaGlnThrPro                              270275280285                                                                  HisThrHisAsnArgThrProGluAsnPheProCysLysAsnLeuAsp                              290295300                                                                     GluAsnTyrCysArgAsnProAspGlyLysArgAlaProTrpCysHis                              305310315                                                                     ThrThrAsnSerGlnValArgTrpGluTyrCysLysIleProSerCys                              320325330                                                                     AspSerSerProValSerThrGluGlnLeuAlaProThrAlaProPro                              335340345                                                                     GluLeuThrProValValGlnAspCysTyrHisGlyAspGlyGlnSer                              350355360365                                                                  TyrArgGlyThrSerSerThrThrThrThrGlyLysLysCysGlnSer                              370375380                                                                     TrpSerSerMetThrProHisArgHisGlnLysThrProGluAsnTyr                              385390395                                                                     ProAsnAlaGlyLeuThrMetAsnTyrCysArgAsnProAspAlaAsp                              400405410                                                                     LysGlyProTrpCysPheThrThrAspProSerValArgTrpGluTyr                              415420425                                                                     CysAsnLeuLysLysCysSerGlyThrGluAlaSerValValAlaPro                              430435440445                                                                  ProProValValLeuLeuProAspValGluThrProSerGluGluAsp                              450455460                                                                     CysMetPheGlyAsnGlyLysGlyTyrArgGlyLysArgAlaThrThr                              465470475                                                                     ValThrGlyThrProCysGlnAspTrpAlaAlaGlnGluProHisArg                              480485490                                                                     HisSerIlePheThrProGluThrAsnProArgAlaGlyLeuGluLys                              495500505                                                                     AsnTyrCysArgAsnProAspGlyAspValGlyGlyProTrpCysTyr                              510515520525                                                                  ThrThrAsnProArgLysLeuTyrAspTyrCysAspValProGlnCys                              530535540                                                                     AlaAlaProSerPheAspCysGlyLysProGlnValGluProLysLys                              545550555                                                                     CysThrThrLysIleLysProArgIleValGlyGlyCysValAlaHis                              560565570                                                                     ProHisSerTrpProTrpGlnValSerLeuArgThrArgPheGlyMet                              575580585                                                                     HisPheCysGlyGlyThrLeuIleSerProGluTrpValLeuThrAla                              590595600605                                                                  AlaHisCysLeuLysLysSerProArgProSerSerTyrLysValIle                              610615620                                                                     LeuGlyAlaHisGlnLysValAsnLeuGluProHisValGlnGluIle                              625630635                                                                     GluValSerArgLeuPheLeuGluProThrArgLysAspIleAlaLeu                              640645650                                                                     LeuLysLeuSerSerProAlaValIleThrAspLysValIleProAla                              655660665                                                                     CysLeuProSerProAsnTyrValValAlaAspArgThrGluCysPhe                              670675680685                                                                  IleThrGlyTrpGlyGluThrGlnGlyThrPheGlyAlaGlyLeuLeu                              690695700                                                                     LysGluAlaGlnLeuProValIleGluAsnLysValCysAsnArgTyr                              705710715                                                                     GluPheLeuAsnGlyArgValGlnSerThrGluLeuCysAlaGlyHis                              720725730                                                                     LeuAlaGlyGlyThrAspSerCysGlnGlyAspSerGlyGlyProLeu                              735740745                                                                     ValCysPheGluLysAspLysTyrIleLeuGlnGlyValThrSerTrp                              750755760765                                                                  GlyLeuGlyCysAlaArgProAsnLysProGlyValTyrValArgVal                              770775780                                                                     SerArgPheValThrTrpIleGluGlyValMetArgAsnAsn                                    785790795                                                                     __________________________________________________________________________

We claim:
 1. A product containing a plasminogen activator and athrombin-activatable plasminogen analogue, for simultaneous orsequential use in thrombolytic therapy.
 2. A method of treating apatient in need of thrombolytic or antithrombotic therapy, whichcomprises the simultaneous or sequential intravenous administration ofeffective amounts of a plasminogen activator and a thrombin-activatableplasminogen analogue.
 3. A product as claimed in claim 1 or a method asclaimed in claim 2 wherein the plasminogen activator is tPA.
 4. Aproduct as claimed in claim 1 or a method as claimed in claim 2 whereinthe thrombin-activatable plasminogen analogue has a cleavage sitesequence P4-P3-Pro-Arg-P1'-P2' where P3 is a basic amino acid residue,P4 is a hydrophobic amino acid residue and each of P1' and P2' isindependently a non-acidic amino acid residue, said site being cleavableby thrombin between Arg and P1'.
 5. A product or a method or a use asclaimed in claim 4, wherein the thrombin-activatable plasminogenanalogue for use in the invention is BB-10153 (SEQ ID.1).